GROUP 8

Luber, Patrishia Anne M.

Obispo, Alejandria G.

2BIOLOGY1
Medina, Ma. Beatrix DL.
Perez, Cristine P.

Exercise 3
Preparation and Sterilization of Culture Media and Glass wares
I.

Introduction
Microorganisms are generally found in our environment as mixed
populations. To be able to know and study specific microorganisms,
scientists must isolate their microscopic species to grow in a pure culture.
Pure culture involves the maintenance of a single species of
microorganisms in a certain environment.
In order to achieve pure cultures of species in the laboratory, Culture
Medias and sterilization methods are used.
Sterilization is one of the hallmarks of successful production of pure
cultures in microbiology. To achieve sterility, it is mandatory to use sterile
equipments and be able to use sterile techniques. Sterilization is the
process of removing all life forms from the glass wares that will be used in
pure culture of microorganisms. Equipments used in pure culture are
subjected to Dry (hot air) or Moist (wet heat) sterilization.
The survival and continued growth of microorganisms depends on the
adequate supply of nutrients from their environment. A solution
containing these nutrients is called Culture Media. All culture medias
may be liquid, semi-solid or solid. A liquid culture media that lacks a
solidifying agent is called a broth. On the other hand, a culture that
contains a solidifying agent called agar, may be semi solid or solid,
depending on the %agar content in the media preparation. Agar is an
extract from seaweed, it contains a complex carbohydrate galactose. It
serves as an excellent solidifying agent because it liquefies at 100 C and
solidifies at around 37 C to 40 C. Because of these properties,
microorganisms could be easily cultivated and cultured. A solid culture
medium requires an agar concentration of around 1.5 -1.8% while a
concentration of less than 1% agar results in a semi-solid culture.

II.

Objectives

III.

To acquire and develop the skills in the preparation of media for the
cultivation of microorganisms
To learn the proper operation of the autoclave and the dry-air oven for
the sterilization of culture media and glass wares

Materials

Culture Media
o

Nutrient Agar (NA)

Nutrient Broth (NB)

Potato Dextrose Agar (PDA)

Other Materials

IV.

Petri dishes

Test tubes

Erlenmeyer flask

Calculator

Serological pipettes

Alcohol lamp

Aluminum foil

Triple beam balance

Procedure
Before preparing the medium, the instructions on the label was read in
order to know how much of the powdered media will be used. The
appropriate amount of NA, NB or PDA was computed and resuspended in
distilled water. After calculating the required amount of the powdered
media, it was weighed in an aluminum foil boat using the triple beam
balance. Next, the weighed powder media was transferred into an
Erlenmeyer flask and the appropriate amount of distilled water was
added. After this, the agar based medium was dissolved by heating
through the use of hotplate, water bath or microwave. For the broth
media, heating was not done because the powdered media was able to
dissolve completely by stirring. Next, the media was transferred onto test
tubes by the use of serological pipettes and cotton plugs were placed on
the test tubes and flasks.

After preparing the media, the glass wares and media were sterilized
separately at121C for 15 minutes in the autoclave. Then, the tubes for
slants were cooled in an inclined position until the medium solidified and
for the deeps, the tubes were cooled in an upright position. For the plated
media, the flasks were cooled to around 45-50C and then were
aseptically poured into a sterile Petri dish and were allowed to solidify.

V.

Data and Results

VI.

Analysis of Data

VII.

Conclusion

VIII.

References

Fox, D. (Eds). (1999). Microbiology Laboratory Manual. Menlo Park, CA:

Benjamin/Cummings Science Publishing